JPWO2019159524A1 - Imaging system and biological object moving device - Google Patents

Abstract

The imaging system includes a camera unit (5) capable of performing a first imaging of the cells (C) before the moving work and a second imaging of the cells after the moving work. From the image acquired in the first imaging, the first determination to determine whether to select cells based on the selection criterion data, and whether to select cells from the image acquired in the second imaging. When the determination unit (781) that executes the second determination to be determined, the storage unit (75) that stores the selection reference data, and the first determination and the second determination have different determination results, A correction unit (782) that updates the selection criterion data stored in the storage unit (75) so that the first determination and the second determination for the cell are the same in the subsequent determination. Be prepared.

Description

The present invention relates to an imaging system including an imaging device for imaging a biological object such as a cell or a cell mass, and a biological object moving device using the imaging system.

For example, in medical or biological research applications, imaging may be performed to screen cells or cell masses (examples of biological objects; sometimes simply referred to as "cells"). For example, the cells contained in the first container of the migration source are imaged with an imaging device, desired cells are selected based on the obtained image, and the selected cells are sucked with a chip into the second container of the migration destination. The work of reprinting may be performed (for example, Patent Document 1).

Specific examples of the cell selection performed on the first container side include a method based on manual selection by the operator, a method of presetting selection reference values related to cell size, shape, and the like. The former is a method that depends on the individual skill that the operator observes the captured image of the cell and makes a pass / fail judgment based on the experience of the operator. The latter is a method in which a parameter relating to the size and shape of the cell is obtained by image processing the captured image of the cell, and the quality determination is automatically determined based on whether or not this parameter satisfies the selection reference value.

As described above, when the cells are selected as desired cells on the first container side of the migration source and the cells migrated to the second container of the migration destination are observed again on the second container side, there are cases where the cells are not intended. obtain. In other words, when the cells are re-observed after the intervention of the "work" of cell migration, they may not actually be the intended cells. The same thing can happen with the intervention of various tasks. For this reason, there is a problem that the work efficiency of inspections and tests performed on the second container side is lowered.

International Publication No. 2015/087371

An object of the present invention is to provide an imaging system capable of accurately selecting a biological object desired by an operator, and a biological object moving device using the imaging system.

The imaging system according to one aspect of the present invention includes a first imaging that images a biological object before a predetermined operation is performed, and a second imaging that images the biological object after the operation is performed. The first determination for determining whether or not to select the biological object based on a predetermined selection criterion from the feasible imaging device and the image acquired in the first imaging, and the acquisition in the second imaging. From the image, a determination unit that executes a second determination for determining whether or not to select the biological object, a storage unit that stores data related to the selection criteria, and the first determination and the second determination Correction to update the data stored in the storage unit so that the first determination and the second determination for the biological object are the same in the subsequent determination when different determination results are obtained. It is characterized by having a part and.

The biological object moving device according to another aspect of the present invention picks a biological object selected as a moving target from the above-mentioned imaging system and a first container accommodating a plurality of biological objects, and then picks a biological object selected as a moving target in the second container. It is provided with a head device that executes the moving work of moving to the predetermined work as the predetermined work.

FIG. 1 is a diagram schematically showing a configuration example of a cell transfer device to which the imaging system according to the embodiment of the present invention is applied. FIG. 2 (A) is a top view of a dish provided in the sorting container used for the cell transfer device, and FIG. 2 (B) is a sectional view taken along line IIB-IIB of FIG. 2 (A). FIG. 3A is a perspective view of a microplate used in the cell transfer device, and FIG. 3B is a vertical sectional view of FIG. 3A. FIG. 4 is a diagram for explaining the concept of feedback of cell selection in the present embodiment. FIG. 5 is a diagram showing a feedback example of cell selection in the case where the work is the movement of cells in the first embodiment. FIG. 6 is a diagram showing a feedback example of cell selection in the second embodiment in which the work is cell selection by an operator. FIG. 7 is a diagram showing a feedback example of cell selection in the third embodiment in which the work is a change in imaging conditions. FIG. 8 is a modified example of the third embodiment, and is a diagram showing a feedback example of cell selection when the work is a change of imaging conditions. FIG. 9 is a diagram showing an example of feedback on cell selection in the fourth embodiment in which the work is dispensing of reagents. FIG. 10 is a diagram for explaining a feedback example of cell selection in the fifth embodiment, in which the work is a work of waiting for the elapse of the test time. FIG. 11 is a block diagram of a cell transfer device according to an embodiment of the present invention. FIG. 12 is a flowchart of a cell movement operation using an imaging system.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the imaging system according to the present invention, a wide variety of biological objects can be imaged. As a biological object to be imaged in the present invention, a cell derived from a living body can be typically exemplified. The cells derived from the living body here are, for example, single cells (cells) such as blood cell lines and single cells, tissue fragments such as Histoculture and CTOS, cell aggregates such as spheroids and organoids, zebrafish, nematodes, and fertilized eggs. Such as individuals, 2D or 3D colonies and the like. In addition, as biological objects, tissues, microorganisms, small-sized species and the like can be exemplified. In the embodiment described below, an example is shown in which the biological object is a cell or a cell aggregate in which several to hundreds of thousands of cells are aggregated (hereinafter, these are collectively simply referred to as “cell C”). ..

[Overall configuration of cell transfer device]
FIG. 1 is a diagram schematically showing an overall configuration of a cell transfer device S to which the imaging system according to the embodiment of the present invention is applied. Here, a cell transfer device S for moving cells C between two containers (dish 2 and microplate 4) is illustrated.

The cell transfer device S includes a translucent base 1 having an upper surface which is a horizontal mounting surface, a camera unit 5 (imaging device) arranged on the lower side of the base 1, and an upper side of the base 1. Includes a head unit 6 (head device) arranged in. A sorting container 11 provided with a dish 2 (first container) is placed in the first mounting position P1 of the base 1, and a microplate 4 (second container) is placed in the second mounting position P2. Has been done. The head unit 6 is equipped with a chip 12 for sucking and discharging cells C, and includes a plurality of heads 61 that can move along the Z direction (vertical direction). The camera unit 5 and the head unit 6 can be moved in the X direction (horizontal direction) and in the direction perpendicular to the paper surface of FIG. 1 (Y direction). The dish 2 and the microplate 4 are placed on the upper surface of the base 1 within the movable range of the head unit 6.

Roughly speaking, the cell transfer device S sucks the cells C individually from the dish 2 of the sorting container 11 holding a large number of cells C to each of the plurality of chips 12, moves the cells C to the microplate 4, and the cells C are transferred to the microplate 4. It is a device that simultaneously ejects cells C from a plurality of chips 12 into wells 41 of a microplate 4. Prior to the suction of the cells C, the cells C held in the dish 2 are imaged (first imaged) by the camera unit 5, and a selection operation of selecting good quality cells C to be moved to the microplate 4 is performed. Will be done. After the movement of the cell C, the cell C housed in the microplate 4 is imaged (second imaging) by the camera unit 5, and the cell C is observed including the purpose of verifying the validity of the selection operation.

Hereinafter, each part of the cell transfer device S will be described. The base 1 is a rectangular flat plate having a predetermined rigidity, and a part or all of the base 1 is made of a translucent material. A preferred base 1 is a glass plate. By forming the base 1 with a translucent material such as a glass plate, the camera unit 5 arranged below the base 1 is combined with the sorting container 11 and the dish 2 arranged on the upper surface of the base 1. The microplate 4 can be imaged through the base 1.

The sorting container 11 is a container that is a source of movement of cells C, stores the medium L, and holds the dish 2 for cell sorting in a state of being immersed in the medium L. The dish 2 is a plate for holding cells C, and has a plurality of holding recesses 3 on the upper surface capable of individually storing and holding cells C. The medium L is not particularly limited as long as it does not deteriorate the properties of the cells C, and can be appropriately selected depending on the type of the cells C.

The sorting container 11 is provided with a rectangular upper opening 11H on the upper surface side thereof. The upper opening 11H is an opening for inputting cells C and picking up selected cells C. The dish 2 is arranged below the upper opening 11H. As the sorting container 11 and the dish 2, those made of a translucent resin material or glass are used. This is because the camera unit 5 arranged below the sorting container 11 makes it possible to observe the cells C supported on the dish 2.

A plurality of cells C dispersed in the cell culture medium are injected into the sorting container 11 from the dispensing chips (not shown). The dispensing chip sucks the cell culture solution together with the cells C from a container for storing the cell culture solution containing a large amount of cells C, and holds the cell culture solution in the dispensing chip. After that, the dispensing tip is moved to an empty position above the sorting container 11 and accesses the upper surface of the dish 2 through the upper opening 11H. Then, with the tip opening of the dispensing chip immersed in the medium L of the sorting container 11, the cells C held in the dispensing chip are discharged onto the dish 2 together with the cell culture solution.

The microplate 4 is a container to which the cells C move, and has a plurality of wells 41 from which the cells C are discharged. The well 41 is a bottomed hole opened on the upper surface of the microplate 4. One well 41 contains a required number (usually one) of cells C together with medium L. As the microplate 4, one made of a translucent resin material or glass is also used. This is because the camera unit 5 arranged below the microplate 4 makes it possible to observe the cells C supported on the well 41.

The camera unit 5 captures an image of cells C held in the sorting container 11 or the microplate 4 from the lower surface side thereof, and includes a lens unit 51 and a camera body 52. The lens unit 51 is an objective lens used in an optical microscope, and includes a lens group for forming an optical image having a predetermined magnification and a lens barrel accommodating the lens group. The camera body 52 includes an image sensor such as a CCD image sensor. The lens unit 51 forms an image of an image of an imaging object on the light receiving surface of the image sensor. The camera unit 5 can move in the X direction and the Y direction below the base 1 along the guide rail 5G extending in the left-right direction in parallel with the base 1. Further, the lens unit 51 can move in the Z direction for focusing operation.

The head unit 6 picks cells C selected as a movement target from a dish 2 as a first container accommodating a plurality of cells C and moves them to a microplate 4 as a second container (predetermined work). ) Is provided, and includes a plurality of heads 61 and a head body 62 to which these heads 61 are assembled. A chip 12 for sucking (picking up) and discharging cells C is attached to the tip of each head 61. The head body 62 holds the head 61 so as to be able to move up and down in the + Z and −Z directions, and can move in the + X and −X directions along the guide rail 6G. The head body 62 can also be moved in the Y direction.

[Details of dish and microplate]
First, the detailed structure of the dish 2 which is the moving source container will be described. FIG. 2A is a top view of the dish 2, and FIG. 2B is a sectional view taken along line IIB-IIB of FIG. 2A. The dish 2 includes a dish body 20 and a plurality of holding recesses 3 formed in the dish body 20. The dish body 20 is made of a flat plate-shaped member having a predetermined thickness, and has an upper surface 21 and a lower surface 22. The holding recess 3 has an opening 31 on the upper surface 21 side, which serves as a receiving opening for cells C. The dish 2 is immersed in the medium L in the sorting container 11. Specifically, the upper surface 21 of the dish body 20 is immersed in the medium L in the sorting container 11, while the lower surface 22 is held in the sorting container 11 at a distance from the bottom plate of the sorting container 11. (See FIG. 1).

Each of the holding recesses 3 includes an opening 31, a bottom 32, a tubular wall surface 33, a hole 34 and a boundary 35. In this embodiment, an example is shown in which square holding recesses 3 are arranged in a matrix in a top view. As shown in FIG. 2B, the plurality of holding recesses 3 are matrix-arranged at a predetermined recess arrangement pitch.

The opening 31 is a square opening provided on the upper surface 21, and has a size that allows the tip opening t of the sorting chip 12 to enter. The bottom 32 is inside the dish body 20 and is located near the bottom 22. The bottom portion 32 is an inclined surface that gently inclines downward toward the center (the center of the square). The tubular wall surface 33 is a wall surface extending vertically downward from the opening 31 toward the bottom portion 32. The hole 34 is a through hole that vertically penetrates between the center of the bottom 32 and the lower surface 22. The boundary portion 35 is located on the upper surface 21 and is a portion serving as an opening edge of each holding recess 3, and is a ridge line that partitions the holding recesses 3 with each other.

The bottom portion 32 and the tubular wall surface 33 of each holding recess 3 partition a storage space 3H for accommodating cells C. Containment space 3H is generally intended to contain one cell C. The hole 34 is provided to allow small cells and impurities other than the desired size to escape from the accommodation space 3H. Therefore, the size of the pore 34 is selected so that cells C of a desired size cannot pass through and small cells and contaminants other than the desired size can pass through. As a result, the cells C to be sorted are trapped in the holding recess 3, while impurities and the like fall from the holes 34 to the bottom plate of the sorting container 11.

Next, the microplate 4 which is the destination container will be described. FIG. 3A is a perspective view of the microplate 4, and FIG. 3B is a vertical sectional view of the microplate 4. The microplate 4 includes a plate body 40 and a plurality of wells 41 arranged in a matrix on the plate body 40. Since the tip opening t of the chip 12 enters the well 41 when the cells C are discharged, each well 41 has an opening diameter that allows the chip 12 to enter with a margin.

There is a standard size for commercially available microplates. The reference microplate has a predetermined length x width size (length 85.48 mm x width 126 mm) and has a predetermined number of wells. The general number of wells is 24 × 16 (384 wells), and these wells are arranged in a matrix at a predetermined pitch. FIG. 3B shows a cross-sectional view of a 384-well microplate 4. As shown in the figure, 24 wells 41 are arranged at an equal well pitch in the longitudinal direction of the microplate 4 (16 in the lateral direction).

[Outline of cell selection work]
FIG. 4 is a diagram for explaining an outline (steps (A) to (C)) of the cell selection work in the present embodiment. First, in step (A), the camera unit 5 images the dish 2 (first image), and acquires the first image of the cells C supported on the holding recess 3 of the dish 2. In this first image, the cell C in the state before the predetermined work, in the present embodiment, the movement work of the cell C is performed, is captured. Then, from the first image, a first determination is executed to determine which cell C among the cells C captured in the image is selected as the movement target based on a predetermined selection criterion.

The selection criteria are, for example, manual selection criteria internally determined by each operator who executes the cell selection operation based on experience or the like, or data of selection criteria regarding a predetermined cell size, shape, or the like. When relying on the latter, the feature amount of the cell C captured in the first image is obtained by image processing. In this case, the first determination is a determination as to whether or not the obtained feature amount satisfies the data of the selection criterion.

In step (B), work on cell C is performed. In the present embodiment, the cells C selected as the movement target in the first determination are individually picked from the holding recess 3 of the dish 2 using the chip 12 and moved to one well 41 of the microplate 4. It's work. The work on cell C is not limited to the above-mentioned movement work. For example, in addition to the case where the cell selection itself by the operator is the above-mentioned work (the second embodiment described later), the work of changing the imaging conditions of the cell C by the camera unit 5 (the third embodiment), and the reagent for the cell C. Etc. (fourth embodiment), waiting for the elapse of the test time for cells C, and the like (fifth embodiment) can be exemplified.

In step (C), the camera unit 5 takes an image (second image) of the microplate 4 to which the cell C has been moved by the moving operation of step (B), and captures the second image of the cell C held in the well 41. get. In the second image, the cells C in the state after the predetermined work is performed are captured. In this second image, a second determination is performed to confirm the validity of each cell C transferred to well 41. In this second determination, for example, which cell C is selected from the cells C captured in the image as the target of the work on the cell C in the next stage, for example, the work such as addition, inspection, and observation of the reagent. It is a judgment of whether or not.

In this way, the cell C selection determination is performed in the two stages of the first determination and the second determination for the following reasons. First, in the work executed between the first and second determinations, that is, the movement work, deformation, alteration, collapse, etc. of the cell C may occur. For example, easily deformable cells C may be deformed when sucked by the chip 12, or what was visible as one cell C may be decomposed into a plurality of cells.

Next, it is possible to make an erroneous determination in the first determination. The first determination is made based on an image of cells C supported on the holding recess 3. That is, the first determination is made based on the feature quantities such as the shape and color tone of the cells C appearing in the first image acquired through the bottom surface of the sorting container 11 and the bottom portion 32 of the holding recess 3. Therefore, an error factor may intervene in the transmitted light image of the cell C, and the lower half surface of the cell C can be observed. Therefore, when the cell C is observed again based on the second image acquired after the work, it may actually be found to be a defective cell C. Further, the second imaging may be recognized as a defective cell C only after the second imaging is performed under different imaging conditions from the first imaging. In addition, by acquiring the second image after performing some treatment on the cell C or setting an aging period, it may be found to be a defective cell C. For this reason, the execution of the second determination is requested.

If a determination result different from that of the first determination is obtained in the second determination in step (C), the discrepancy is fed back to the first determination in the next procedure (A). Specifically, if a discrepancy cell C (error) in which different determinations are made in the first and second determinations occurs, the feature amount of the cell C (error) can be obtained from, for example, the second image. Then, in the subsequent determination process, the selection criterion data on which the first determination depends is used as the cell C (error) so that the first determination and the second determination for the cell C (error) are the same. Update by referring to the features of. By repeating such updating (learning), only the cells C determined to be "selected" in the second determination are selected in the first determination, and the first determination and the second determination are made. Is gradually homogenized. Eventually, the truly necessary cell C can be selected only by the first determination, the second determination can be omitted, and the work efficiency can be improved. Hereinafter, an example of feedback on cell selection is specifically shown for each work type.

[First Embodiment]
FIG. 5 is a diagram schematically showing feedback procedures (A) to (G) of cell selection in the case where the work is the movement of cells, which has been outlined above in the first embodiment. Is. In the first step (A), the first imaging in which the camera unit 5 images the dish 2 is executed. By this first imaging, the first image of the cell C supported on the holding recess 3 of the dish 2 is acquired. The lens unit 51 of the camera unit 5 has an angle of view capable of simultaneously capturing a plurality of cells C. In the procedure (A), an example is shown in which the holding recess 3 of the matrix of 3 × 3 (m1 to m3 × n1 to n3) is imaged by one imaging operation. Here, one cell C is supported in the holding recesses 3 of m1n3 and m2n3, two cells C are held in the holding recesses 3 of m2n2, m3n1 and m3n3, and cells are held in the other holding recesses 3. Indicates a state in which C is not held.

In the subsequent procedure (B), the first determination as to which cell C is to be moved is executed based on the first image acquired in the procedure (A). As described above, the selection criterion data is used for this first determination. By analyzing the first image, the feature amount of the cell C held in each holding recess 3 is extracted. The feature amount is, for example, the amount of cell C obtained from the number, area, estimated volume, etc. of cell C, the color and pattern of cell C, the light intensity when the cell C is fluorescent, and the like. The analysis result of the cell C in each holding recess 3 is quantified. The selection criterion data is, for example, a parameter that defines the range of cells C to be selected. In the first determination, it is determined whether or not the feature amount of each cell C belongs to the above parameter range, and the belonging cell C is selected as a migration target. Here, an example in which the cell C1 of m1n3 and the cell C2 of m2n3 are selected as migration targets is shown (in FIG. 5, it is emphasized by a black frame. The same applies hereinafter).

In the next step (C), as a predetermined operation, the cells C1 and C2 selected as the migration targets are transferred by the chip 12 to each well 41 of the microplate 4. When the movement is completed, as shown in the procedure (D), the camera unit 5 performs a second imaging in which the microplate 4 holding the moved cells C1 and C2 is imaged. Then, based on the second image obtained by this second imaging, the second determination as to whether or not to select the cells C1 and C2 for the subsequent work is executed. This second determination may be executed by either manual selection by the operator or automatic selection using the feature amount. Here, an example is shown in which cell C1 is selected (OK) and cell C2 is not selected (NG) in the second determination. As the feature amount, information such as the number, amount, color, pattern, and light intensity of the cells C housed in the well 41 can be used. In particular, since the number of cells C is the most easily identifiable information including the case of 0 cells, it is desirable to include it in the feature amount at the minimum.

In the subsequent procedure (E), the feature amount of the cell C1 selected in the second determination or the cell C2 not selected in the second determination is extracted based on the second image. Then, as shown in the procedure (F), the extracted feature amount is fed back (updated) to the selection reference data used for the first determination. As a result, in the first determination based on the conventional selection criterion data, the cell C2 is determined to be "selected", but in the subsequent first determination, the cell C2 is determined to be "not selected". ..

Procedure (G) shows an example of cell selection after feedback. That is, in the first determination after feedback, cell C1 or a cell C in a category similar thereto is selected, but cell C2 or a cell C in a category similar thereto is not selected. After that, the process proceeds to the procedure (C), the selected cells C1 are moved to each well 41, and the second determination of the procedure (D) is executed in the same manner. In this case, since only cell C1 will be transferred to well 41, these will be selected in the second determination. That is, in the selection of cell C, there is no discrepancy between the first determination and the second determination.

[Second Embodiment]
FIG. 6 is a diagram showing a feedback example of cell selection in the second embodiment in which the work is cell selection by an operator. Here, an example is shown in which the operator manually reselects the validity of the cells C selected in the first determination based on the above-mentioned selection criterion data in the dish 2. That is, before the movement work of the cell C described in the first embodiment, the operator executes the work of the manual selection operation that also serves as the second determination for the cell C automatically selected based on the selection reference data. Then, the result of the cell C by the manual selection operation is fed back to the first determination.

FIG. 6A shows the result of the first determination made on the first image acquired by the first imaging based on the selection reference data at a certain time point. Here, an example in which the m1n3 cell C1 and the m2n3 cell C2 are selected in the first determination is shown. The operator performs a second determination (reselection operation) as to whether or not these cells C1 and C2 are appropriate as targets to be selected by a manual selection operation. Since the image of the dish 2 is referred to in the second determination, the first image acquired earlier is used. That is, in the present embodiment, the second image is not newly acquired, and the first image is used as the second image. The present invention also includes such embodiments.

FIG. 6B is a diagram showing the result of the second determination by the operator. Here, an example is shown in which cell C1 was selected in the second determination, but cell C2 was not selected. After that, the feature amount of the cell C1 or the cell C2 is extracted in the same manner as in the procedures (E) and (F) of the first embodiment, and the extracted feature amount is the selection criterion data used for the first determination. Will be fed back to. As a result, in the first determination performed thereafter, the cell C2 is determined to be "not selected".

[Third Embodiment]
FIG. 7 is a diagram showing a feedback example of cell selection in the third embodiment in which the work is a change in imaging conditions. As described above, in the embodiment of the present invention, the first determination and the second determination are executed based on the two-dimensional image of the cell C. In this case, by changing the imaging conditions, it may be possible to clearly determine the quality of the cells C, for which it was difficult to determine the quality of the images captured under the same imaging conditions. In view of this point, in the third embodiment, the first imaging is executed under predetermined imaging conditions to acquire the first image and the first determination is performed, and in the subsequent second imaging, the imaging conditions are changed to obtain the second image. Is acquired and the second determination is made. That is, the work in the third embodiment is the work of changing the imaging conditions in the first imaging. Then, the selection result of the cell C by the second determination is fed back to the first determination.

FIG. 7A is a first image of the cell C acquired by the first imaging, and shows the image obtained by the bright field imaging. That is, it is an image acquired by the camera unit 5 under the condition of "bright field imaging". In this first image, the first determination is executed based on the selection criterion data at that time. After that, the work of changing the imaging conditions is performed. The imaging conditions that can be changed include, for example, the angle of view, the imaging angle, the magnification, the exposure amount, the nature of the illumination light, the amount of light, the lens type, and the like. Of course, the change of the camera unit 5 to be used is also included.

FIG. 7B is a second image of cells C acquired by the second imaging after changing the imaging conditions, and here shows an image obtained by fluorescence imaging. That is, it is an image acquired by the camera unit 5 under the condition of "fluorescence imaging". The fluorescence imaging is performed, for example, by adding a fluorescent agent or using fluorescent illumination. In this second image, for example, the operator makes a second determination as to which cell C is selected by a manual selection operation. The selection result of the cell C by the second determination is fed back to the first determination.

When the m1n3 cells C1a and the m2n3 cells C2a observed in the first image of the bright-field imaging are observed in the second image of the fluorescence imaging, they are observed as cells C1b and C2b of different optical images. Here, the defect of cell C that was overlooked in the bright field imaging may be observed in the fluorescence imaging. That is, there is a case where more accurate selection determination can be performed based on the image acquired by changing the viewpoint of the cell C. Then, the defect may actually appear as a feature amount in the first image of the bright field imaging. Therefore, by feeding back the result of the second determination based on the second image of the fluorescence imaging to the selection reference data, the first determination can be made more accurately.

FIG. 8 is a diagram showing a modified example of the third embodiment. The work of changing the imaging conditions in this modified example is the work of changing the imaging magnification of the lens unit 51 of the camera unit 5. FIG. 8A is a first image of the cell C acquired by the first imaging, and shows, for example, an image captured by a lens having a magnification of 4 times. On the other hand, FIG. 8B is a second image of the cell C acquired by the second imaging after changing the imaging magnification, and shows, for example, an image of the cell C2 of m2n3 imaged with a lens having a magnification of 10 times. ing.

The selection necessity judgment (first judgment) made for the cell C2 based on the first image of FIG. 8 (A) is also the selection required made for the cell C2 based on the enlarged second image of FIG. 8 (B). In some cases, the negative judgment (second judgment) can make a more accurate judgment. For example, a defect that could not be identified in the 4x image may be observable in the 10x image. Therefore, by feeding back the result of the second determination based on the high-magnification second image to the selection reference data, the first determination can be made more accurately.

[Fourth Embodiment]
FIG. 9 is a diagram showing an example of feedback on cell selection in the fourth embodiment in which the work is dispensing of reagents. In many cases, the selected cells C are subjected to a sensitivity test to a reactivity test substance such as a reagent or a growth agent. It is possible that what was selected as good quality cell C suitable for the test subject was cell C unsuitable for the sensitivity test. In view of this point, in the fourth embodiment, after the first determination, the operation of adding the reactivity test substance to the selected cell C is performed, and then the second imaging is performed on the cell C to perform the second imaging. 2 The image is acquired and the quality of the cell C is judged (second judgment). Then, the selection result of the cell C by the second determination is fed back to the first determination.

FIG. 9A shows the result of the first determination made on the first image acquired by the first imaging based on the selection reference data at a certain time point. Here, an example in which the m1n3 cell C1 and the m2n3 cell C2 are selected in the first determination is shown. As the first operation, the selected cells C1 and C2 are moved by the chip 12 to each well 41 of the microplate 4. Up to this point, it is the same as the first embodiment described above.

Subsequently, as shown in FIG. 9B, as a second operation, the reagent Q is dispensed to the wells 41 containing the transferred cells C1, C2 and the medium L, respectively, using the chip 12. Is executed. After a predetermined time has passed after the dispensing of the reagent Q is completed, as shown in FIG. 9C, a second imaging in which the camera unit 5 images the microplate 4 holding the moved cells C1 and C2 is performed. .. As a result, images of cells C1 and C2 after reacting with reagent Q are acquired as the second image. In this second image, for example, the operator makes a second determination by a manual selection operation to select whether or not the cells C1 and C2 are suitable as the target of the sensitivity test. Here, an example is shown in which cell C1 is selected (OK) and cell C2 is not selected (NG) in the second determination.

Then, as shown in FIG. 9D, the selection result of the cells C1 and C2 by the second determination is fed back to the first determination. That is, the feature amount of the cell C1 or the cell C2 is extracted, and the extracted feature amount is fed back (updated) to the selection criterion data used for the first determination. As a result, in the first determination performed thereafter, the cell C2 is determined to be "not selected". Therefore, the cells C are selected before the reagent Q is added so as to be in line with the cell C selection result after the reagent Q is added.

[Fifth Embodiment]
FIG. 10 is a diagram for explaining a feedback example of cell selection in the fifth embodiment, in which the work is a work of waiting for the elapse of the test time. The shape and properties of cell C may be displaced even if it is left in the medium L without adding reagent Q or the like. For example, cell C growth, death, division, discoloration and the like can be exemplified. FIG. 10 shows an example in which the color of cell C changes with the passage of time, and that of cells C (t1), C (t2), and C (t3) after 5 hours, 24 hours, and 36 hours have passed. The state is simply illustrated. Therefore, waiting for the elapse of a predetermined test time for cell C can also be a "work" for cell C.

The fifth embodiment can be carried out, for example, by substituting the reagent dispensing work in FIG. 9B of the fourth embodiment with the work of waiting for the elapse of the test time. In this case, the second imaging and cell selection (second determination) of FIG. 9C are performed after a predetermined test time has elapsed after moving the cells C1 and C2 to the well 41. Then, the result of the second determination is fed back to the first determination.

[Electrical configuration of cell transfer device]
FIG. 11 is a block diagram showing an electrical configuration of the cell transfer device S. The cell moving device S includes a control unit 7 that controls the movement of the head unit 6, the raising and lowering of the head 61 and the chip 12, the suction and discharge operations of the cells C, the movement of the camera unit 5, the imaging operation, and the like. Further, the cell moving device S has a camera shaft drive unit 53 as a mechanism for horizontally moving the camera unit 5, a servomotor 54 as a drive source for moving the lens unit 51 up and down, and a head unit shaft drive as a mechanism for horizontally moving the head unit 6. The head drive unit 64 is provided as a mechanism for raising and lowering the unit 63 and the head 61, and a mechanism for performing suction and discharge operations.

The camera shaft drive unit 53 includes a drive motor that horizontally moves the camera unit 5 along the guide rail 5G (FIG. 1). The camera shaft drive unit 53 moves the camera unit 5 between the first mounting position P1 directly below the dish 2 and the second mounting position P2 directly below the microplate 4.

The servomotor 54 rotates forward or reverse to move the lens portion 51 in the vertical direction with a predetermined resolution via the power transmission mechanism (not shown). By this movement, the focal position of the lens portion 51 is aligned with the cells C housed in the holding recess 3 of the dish 2 or the well 41 of the microplate 4. As shown by the dotted line in FIG. 11, the servomotor 54 moves the sorting container 11 or the microplate 4 itself, or the base 1 on which these are placed, up and down instead of the lens portion 51. You may let it.

The head unit shaft drive unit 63 includes a drive motor that moves the head unit 6 (head body 62) along the guide rail 6G. The head drive unit 64 includes a motor that serves as a power source for raising and lowering the head 61 with respect to the head body 62, and a mechanism that serves as a power source for generating suction force and discharge force at the tip opening t of the chip 12.

The control unit 7 is composed of a microcomputer or the like, and when a predetermined program is executed, the axis control unit 71, the head control unit 72, the image pickup control unit 73, the image processing unit 74, the storage unit 75, and the main control unit 78 are combined. Functions to prepare. Further, the control unit 7 is provided with an input unit 76 for inputting various information and a display unit 77 for displaying various information. The input unit 76 functions as a terminal that receives an input related to the cell C selection operation from the operator. The display unit 77 functions as a monitor for displaying the first image, the second image, and the like captured by the camera unit 5.

The shaft control unit 71 controls the operation of the head unit shaft drive unit 63. That is, the shaft control unit 71 moves the head unit 6 to a predetermined target position in the horizontal direction by controlling the head unit shaft drive unit 63. The movement of the head 61 (tip 12) between the sorting container 11 and the microplate 4, the positioning of the dish 2 with respect to the holding recess 3 above the vertical, and the positioning of the head 61 (chip 12) with respect to the well 41 of the microplate 4 to be discharged. Positioning and the like are realized by the control of the head unit shaft drive unit 63 by the shaft control unit 71.

By controlling the head drive unit 64, the head control unit 72 raises and lowers the head 61 to be controlled toward a predetermined target position. Further, the head control unit 72 controls a suction mechanism corresponding to the head 61 to be controlled to generate a suction force or a discharge force at the tip opening t of the chip 12 at a predetermined timing.

The image pickup control unit 73 controls the camera shaft drive unit 53 to control the operation of moving the camera unit 5 along the guide rail 5G. Further, the image pickup control unit 73 controls the image pickup operation of the dish 2 or the microplate 4 by the camera unit 5, such as the exposure amount and the shutter timing. Further, the imaging control unit 73 gives the servomotor 54 a control pulse for moving the lens unit 51 in the vertical direction at a predetermined pitch (for example, a pitch of several tens of μm) for the focusing operation.

The image processing unit 74 performs image processing such as edge detection processing and pattern recognition processing accompanied by feature amount extraction on the image data acquired by the camera body 52. The image processing unit 74 recognizes the presence and number of cells C on the holding recess 3 of the dish 2 on the image based on the image of the dish 2 after the cells C have been dispensed, and the XY of each cell C. A process of acquiring coordinates, a process of acquiring condition information such as the outer contour of each cell C, a size such as an area and a volume, a shape, and a color tone are executed. Similarly, the image processing unit 74 performs a process of recognizing the number of cells C housed in the well 41, the total area, the total volume, etc., the fluorescence intensity, etc., based on the image of the well 41 to which the cells C have been moved. Execute.

The storage unit 75 stores various set values, data, programs, and the like in the cell moving device S. In addition, the storage unit 75 stores data relating to the cell C selection criteria used in the above-mentioned first determination. As described above, this selection criterion data is updated according to the cell selection result of the second determination performed after the predetermined work on the cell C is performed.

The main control unit 78 comprehensively controls the operations of the camera unit 5 and the head unit 6. The main control unit 78 causes the dish 2 in which the cells C are sown to be imaged at the first placement position P1 (FIG. 1) in which the sorting container 11 is placed, and at the same time, the cells C selected as the movement target are selected. The camera unit 5 and the head unit 6 are picked by sucking the chips 12 mounted on the head 61, and the cells C are moved to the microplate 4 through the axis control unit 71, the head control unit 72, and the imaging control unit 73. To control. In such integrated control, the main control unit 78 makes a first determination for automatically selecting cells C to be moved from the dish 2, and whether or not to select cells C after moving to the microplate 4. The second determination for determining whether or not is executed.

The main control unit 78 functionally includes a determination unit 781, a correction unit 782, and an analysis unit 783 for the first and second determinations described above. The determination unit 781 executes the first determination process of selecting the cells C to be moved to the microplate 4 from the first image of the dish 2 carrying the cells C imaged (first imaged) by the camera unit 5. To do. The determination unit 781 refers to the selection reference data stored in the storage unit 75, and the feature amount of each cell C acquired by the image processing of the first image by the image processing unit 74 matches the selection reference data. The first determination is executed based on whether or not.

Further, the determination unit 781 performs a second determination process of selecting the cell C to be used for the subsequent operation from the second image of the microplate 4 after the movement operation of the cell C captured by the camera unit 5 (second imaging). To execute. This second determination can depend on the operation of selecting cells C from the operator, which is accepted by the input unit 76. Instead of this, the selection reference data for the second determination may be stored in the storage unit 75, and the determination unit 781 may automatically determine.

When the first determination and the second determination result in different determination results for a certain cell C, the correction unit 782 determines that the first determination and the second determination for the cell C are different in the subsequent determination. A process of updating the selection reference data stored in the storage unit 75 is performed so that the data is the same. Specifically, the correction unit 782 reflects the characteristic amount of the inconsistent cell C in the selection criterion data. That is, when the cell C is "non-selected" in the second determination, the selection criterion data is updated so that the feature amount of the cell C is not included in the selection criterion data. On the other hand, when the cell C that was "non-selected" in the first determination is "selected" in the second determination, the feature amount of the cell C is conversely included in the selection criterion data. The selection criterion data is updated.

The analysis unit 783 performs a process of extracting the feature amount of the cell C by analyzing the image of the cell C specified by the image processing unit 74 in the above-mentioned first image or the second image. The feature amount to be extracted is, for example, the shape, number, area, estimated volume, color, pattern, light intensity, etc. of cell C. The analysis unit 783 digitizes these feature quantities, and the determination unit 781 and the correction unit 782 perform a predetermined process using these numerical values. For example, the analysis unit 783 extracts the feature amount of the cell C selected in the second determination or the cell C not selected in the second determination based on the image acquired in the second imaging. The correction unit 782 updates the selection reference data by using the numerical value of the extracted feature amount.

The determination unit 781 executes an automatic determination mode in which the result of the first determination is used for the second determination at the stage where the selection reference data is determined to have progressed in learning by the update process of the correction unit 782. It is possible. By repeating the update (learning) of the selection criterion data, the first determination and the second determination are gradually homogenized, and the truly necessary cell C can be selected only by the first determination. Because.

[Flow of cell migration movement]
Subsequently, the cell movement operation using the imaging system of the present embodiment shown in FIG. 11 will be described with reference to the flowchart shown in FIG. When the process is started, the main control unit 78 causes the camera unit 5 to image the dish 2 (first image). A cell suspension is dispensed into the dish 2 in advance, and the camera unit 5 images a state in which the cells C are contained in the holding recess 3 of the dish 2 (step S1).

Next, the image processing unit 72 acquires the image data of the dish 2 acquired by the imaging from the camera body 52, and executes image processing for identifying the cells C contained in the image. This image processing data is sent to the analysis unit 783 of the main control unit 78, and the analysis unit 783 has the shape, number, area, estimated volume, color, pattern, light intensity, etc. of the identified cell C. The process of obtaining the feature amount of is executed (step S2).

Subsequently, the determination unit 781 reads out the selection reference data of the cell C from the storage unit 75 (step S3), refers to the selection reference data, and moves any of the cell C carried on the dish 2. The first determination for determining whether or not to select as a target is executed (step S4).

Once the cells C to be migrated are determined, the main control unit 78 executes a cell migration operation of moving the cells C from the dish 2 first container as the first container to the microplate 4 as the second container (step S5). .. Specifically, the head control unit 72 controls the head drive unit 64 to cause the chip 12 mounted on the head 61 to pick the cells C supported on the holding recess 3 of the dish 2. Further, the shaft control unit 71 controls the head unit shaft drive unit 63 to move the head unit 6 to the sky above the microplate 4. Further, the head control unit 72 controls the head drive unit 64 to discharge the cells C sucked by the chip 12 to a predetermined well 41.

When the discharge of the cells C to the well 41 is completed, the main control unit 78 causes the camera unit 5 to take an image (second image) of the microplate 4 (step S6). The image acquired by this imaging is displayed on the display unit 77. The operator visually recognizes the display unit 77, and determines which cell C is selected for the subsequent work among the cells C moved to each well 41 of the microplate 4. The determination result is accepted by the input unit 76. The determination unit 781 treats the instruction information input to the input unit 76 as a second determination for selecting the cell C after a predetermined operation (step S7).

If there are cells C for which different determinations have been made in the first determination in step S4 and the second determination in step S7, the feature amount of the cell C is calculated by the analysis unit 783 (step S8). Then, the correction unit 782 updates the selection reference data for the first determination stored in the storage unit 75 based on the feature amount extracted by the analysis unit 783 (step S10). If there is no discrepancy between the first determination and the second determination, steps S8 and S9 are skipped.

Subsequently, the main control unit 78 determines whether or not the operation mode set in the cell movement device S is the manual operation mode (step S10). The manual operation mode is a mode in which imaging in step S6 and input acceptance in step S7 are executed after the cell C movement operation, and is executed when the selection reference data of the storage unit 75 is not sufficiently learned. Is.

When the manual operation mode is set (YES in step S10), the main control unit 78 confirms whether or not the next dish 2 imaging (first imaging) is scheduled (step S11). When the first imaging is scheduled (YES in step S11), the main control unit 78 returns to step S1 to execute the next first imaging. In the next routine, in step S4, the selection criterion data previously updated in step S9 is used.

On the other hand, in step S10, when the automatic determination mode is set instead of the manual operation mode (NO in step S10), the main control unit 78 executes the operation in which the above steps S6 to S9 are omitted. The automatic determination mode is a mode executed when the learning of the selection reference data is determined to have progressed by the update process of the correction unit 782, and the result of the first determination is the second determination. It is a mode to be treated as the result of.

In this case, the main control unit 78 causes the camera unit 5 to image the dish 2 carrying the cell C (first imaging) (step S12), and the analysis unit 783 calculates the feature amount of the cell C in the acquired image. (Step S13). Subsequently, the determination unit 781 makes a first determination to select the cell C to be moved with reference to the selection criterion data of the storage unit 75 (step S14). Then, the main control unit 78 moves the selected cells C from the dish 2 to the microplate 4 (step S15). As described above, since the second determination is omitted in the automatic determination mode, the next step work, for example, addition of a reagent, is executed for all the transferred cells C.

After that, it is confirmed whether or not to continue imaging the cells C (step S16). When continuing the imaging (YES in step S16), the process returns to step S13, and the camera unit 5 executes the next imaging operation on the dish 2. On the other hand, when there is no cell C to be imaged (NO in step S16), the process ends.

According to the imaging system according to the present embodiment described above, regarding the selection of the cell C, the determination unit 781 is based on the selection criterion data in the image acquired in the first imaging executed before the predetermined work on the cell C. The selected first determination is compared with the second determination based on the image acquired in the second imaging acquired after the predetermined work has been performed. Then, when there is a discrepancy between the two, the correction unit 782 updates the selection reference data so that the first determination and the second determination are the same. Therefore, the selection criterion data is gradually modified so as to match the second determination performed after the predetermined work. Therefore, the probability of determining "selection" in the second determination can be increased, and the work efficiency of subsequent tests and tests on the cell C can be increased.

[Invention included in the above embodiment]
The specific embodiments described above mainly include inventions having the following configurations.

The imaging system according to one aspect of the present invention includes a first imaging that images a biological object before a predetermined operation is performed, and a second imaging that images the biological object after the operation is performed. The first determination for determining whether or not to select the biological object based on a predetermined selection criterion from the feasible imaging device and the image acquired in the first imaging, and the acquisition in the second imaging. From the image, a determination unit that executes a second determination for determining whether or not to select the biological object, a storage unit that stores data related to the selection criteria, and the first determination and the second determination Correction to update the data stored in the storage unit so that the first determination and the second determination for the biological object are the same in the subsequent determination when different determination results are obtained. It is characterized by having a part and.

According to this imaging system, regarding the selection of a biological object, the first determination made based on a predetermined selection criterion in the image acquired by the first imaging and the acquisition after performing a predetermined operation are performed. The second determination based on the image acquired in the second imaging is compared. Then, when there is a discrepancy between the two, the data regarding the selection criterion is updated so that the first determination and the second determination are the same. Therefore, the data of the selection criteria is gradually modified so as to match the second determination performed after the predetermined work. Therefore, it is possible to increase the probability of determining "selection" in the second determination, and it is possible to increase the work efficiency of subsequent inspections and tests on the biological object.

In the above imaging system, the selection operation received by the input unit is further provided with an input unit that receives an input related to the selection operation for the biological object from the operator, and the predetermined work also serves as the second determination. Is desirable.

According to this imaging system, the data of the selection criterion on which the first determination is based is updated so as to be in line with the selection operation result of the operator who also serves as the second determination. That is, the result of the operator's selection operation is fed back to the first determination. Therefore, the result of the first determination gradually follows the tendency of the operator's selection operation, and the second determination (selection operation) can be simplified.

In the above imaging system, the predetermined work is a work of moving a biological object selected as a moving target from a first container accommodating a plurality of biological objects to a second container, and the first imaging is It is desirable that the image of the biological object housed in the first container is taken, and the second imaging is the image of the biological object moved to the second container.

According to this imaging system, the result of the second determination based on the image of the second imaging, which is executed after the work of moving the biological object from the first container to the second container, is fed back to the first determination. Will be done. Therefore, the biological object is selected before the moving work so as to be in line with the selection result of the biological object after the moving work.

In this case, with respect to the biological object selected in the second determination or the biological object not selected, an analysis unit that extracts the feature amount of the biological object based on the image acquired by the second imaging is further added. It is desirable that the correction unit updates the data related to the selection criterion based on the feature amount extracted by the analysis unit.

According to this imaging system, the result of the second determination can be objectively evaluated based on the feature amount, and this can be fed back to the data of the selection criterion.

Further, in this imaging system, the second container may include a plurality of wells for accommodating the biological object, and the feature amount may include information on the number of the biological objects accommodated in each of the wells. desirable. As a result, the number information of the biological object can be reflected in the data of the selection criterion.

In the above imaging system, it is desirable that the predetermined work is an operation of changing the imaging conditions in the first imaging, and the second imaging is executed under the changed imaging conditions.

According to this imaging system, the second determination is made based on the image acquired after changing the imaging conditions, that is, the image acquired by changing the viewpoint of the biological object. Then, the result of this second determination is fed back to the first determination. Therefore, the biological object is selected in the first determination so as to be in line with the selection result of the biological object from which the viewpoint is changed.

In the above imaging system, it is desirable that the predetermined work is the work of adding the reactivity test substance to the biological object.

According to this imaging system, the result of the second determination based on the image of the second imaging, which is executed after the operation of adding the reactivity test substance to the biological object, is fed back to the first determination. Therefore, the biological object is selected before the addition operation so as to be in line with the selection result of the biological object after the addition operation of the reactivity test substance.

In the above imaging system, it is desirable that the predetermined operation is an operation of waiting for the lapse of a predetermined test time after the first imaging.

According to this imaging system, the result of the second determination based on the image of the second imaging, which is executed after the work of waiting for the elapse of a predetermined test time, is fed back to the first determination. Therefore, the biological object is selected in the first determination so as to be in line with the selection result of the biological object after the lapse of the test time.

In these imaging systems, the predetermined work further includes the work of moving the biological object selected as the moving target from the first container accommodating a plurality of biological objects to the second container, and the first imaging. Is an image of a biological object housed in the first container, and the second imaging is an image of a biological object moved to the second container, and is an operation of adding the reactivity test substance or It is desirable that the work of waiting for the elapse of the test time is performed on the biological object after being transferred to the second container.

In the above imaging system, the determination unit automatically uses the result of the first determination for the second determination at the stage where it is determined that learning has progressed due to the update of the correction unit for the data related to the selection criterion. It is desirable to have a determination mode.

According to this imaging system, when it is determined that the learning has progressed by advancing the feedback to the first determination, the second determination is omitted. Therefore, the work efficiency can be further improved.

The biological object moving device according to another aspect of the present invention picks a biological object selected as a moving target from the above-mentioned imaging system and a first container accommodating a plurality of biological objects, and then picks a biological object selected as a moving target in the second container. It is provided with a head device that executes the moving work of moving to the predetermined work as the predetermined work.

According to the present invention, it is possible to provide an imaging system capable of accurately selecting a biological object desired by an operator, and a biological object moving device using the imaging system.

Claims (11)

An imaging device capable of performing a first imaging of an image of a biological object before a predetermined operation is performed and a second imaging of an image of the biological object after the operation is performed.
From the image acquired by the first imaging, the first determination to determine whether or not to select the biological object based on a predetermined selection criterion, and from the image acquired by the second imaging, the biological object. A determination unit that executes a second determination to determine whether or not to select
A storage unit that stores data related to the selection criteria,
When the first determination and the second determination have different determination results, the storage unit so that the first determination and the second determination for the biological object are the same in the subsequent determination. A correction unit that updates the data stored in
An imaging system characterized by being equipped with. In the imaging system according to claim 1,
Further provided with an input unit that receives input related to the selection operation for the biological object from the operator.
An imaging system in which the predetermined operation is the selection operation received by the input unit, which also serves as the second determination. In the imaging system according to claim 1 or 2.
The predetermined work is a work of moving a biological object selected as a moving target from a first container accommodating a plurality of biological objects to a second container.
The first imaging is an imaging of a biological object housed in the first container.
The second imaging is an imaging system in which an imaging of a biological object moved to the second container is performed. In the imaging system according to claim 3,
With respect to the biological object selected in the second determination or the biological object not selected, an analysis unit for extracting the feature amount of the biological object based on the image acquired by the second imaging is further provided.
The correction unit is an imaging system that updates data related to the selection criteria based on the feature amount extracted by the analysis unit. In the imaging system according to claim 4,
The second container comprises a plurality of wells for accommodating the biological object.
An imaging system in which the feature amount includes information on the number of biological objects housed in each of the wells. In the imaging system according to claim 1 or 2.
The predetermined work is a work of changing the imaging conditions in the first imaging.
The second imaging system is an imaging system in which the second imaging is performed under the modified imaging conditions. In the imaging system according to claim 1 or 2.
An imaging system in which the predetermined operation is an operation of adding a reactivity test substance to the biological object. In the imaging system according to claim 1 or 2.
An imaging system in which the predetermined operation is an operation of waiting for the lapse of a predetermined test time after the first imaging. In the imaging system according to claim 7 or 8.
The predetermined work further includes the work of moving the biological object selected as the moving target from the first container accommodating a plurality of biological objects to the second container.
The first imaging is an imaging of a biological object housed in the first container.
The second imaging is an imaging of a biological object moved to the second container.
An imaging system in which the work of adding the reactivity test substance or the work of waiting for the lapse of the test time is performed on a biological object after being moved to the second container. In the imaging system according to any one of claims 1 to 9,
The determination unit has an automatic determination mode in which the result of the first determination is used for the second determination at the stage where it is determined that the learning has progressed due to the update of the correction unit for the data related to the selection criterion. system. The imaging system according to any one of claims 1 to 10.
A head device that picks a biological object selected as a moving target from a first container accommodating a plurality of biological objects and executes a moving operation of moving to the second container as the predetermined work.
A biological object moving device comprising.

Images